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Suk-chul Bae - One of the best experts on this subject based on the ideXlab platform.
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Role of RUNX Family Members in G 1 Restriction-Point Regulation
Molecules and cells, 2020Co-Authors: Jung-won Lee, Suk-chul BaeAbstract:When cells are stimulated by growth factors, they make a critical choice in early G1 phase: proceed forward to S phase, remain in G1, or revert to G0 phase. Once the critical decision is made, cells execute a fixed program independently of extracellular signals. The specific stage at which the critical decision is made is called the Restriction Point or R-Point. The existence of the R-Point raises a major question: what is the nature of the molecular machinery that decides whether or not a cell in G1 will continue to advance through the cell cycle or exit from the cell cycle? The R-Point program is perturbed in nearly all cancer cells. Therefore, exploring the nature of the R-Point decision-making machinery will provide insight into how cells consult extracellular signals and intracellular status to make an appropriate R-Point decision, as well into the development of cancers. Recent studies have shown that expression of a number of immediate early genes is associated with the R-Point decision, and that the decision-making program constitutes an oncogene surveillance mechanism. In this review, we briefly summarize recent findings regarding the mechanisms underlying the context-dependent R-Point decision.
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Involvement of RUNX and BRD Family Members in Restriction Point.
Molecules and cells, 2019Co-Authors: Jung-won Lee, Tae-geun Park, Suk-chul BaeAbstract:A tumor is an abnormal mass of tissue that arises when cells divide more than they should or do not die when they should. The cellular decision regarding whether to undergo division or death is made at the Restriction (R)-Point. Consistent with this, an increasingly large body of evidence indicates that deregulation of the R-Point decision-making machinery accompanies the formation of most tumors. Although the R-Point decision is literally a matter of life and death for the cell, and thus critical for the health of the organism, it remains unclear how a cell chooses its own fate. Recent work demonstrated that the R-Point constitutes a novel oncogene surveillance mechanism operated by R-Point-associated complexes of which RUNX3 and BRD2 are the core factors (Rpa-RX3 complexes). Here, we show that not only RUNX3 and BRD2, but also other members of the RUNX and BRD families (RUNX1, RUNX2, BRD3, and BRD4), are involved in R-Point regulation.
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Runx3 plays a critical role in Restriction-Point and defense against cellular transformation.
Oncogene, 2017Co-Authors: X. Z. Chi, Yoshiaki Ito, J Lee, Y-s Lee, Il Yeong Park, Suk-chul BaeAbstract:The Restriction (R)-Point decision is fundamental to normal differentiation and the G1-S transition, and the decision-making machinery is perturbed in nearly all cancer cells. The mechanisms underlying the cellular context-dependent R-Point decision remain poorly understood. We found that the R-Point was dysregulated in Runx3-/-mouse embryonic fibroblasts (MEFs), which formed tumors in nude mice. Ectopic expression of Runx3 restored the R-Point and abolished the tumorigenicity of Runx3-/-MEFs and K-Ras-activated Runx3-/-MEFs (Runx3-/-;K-RasG12D/+). During the R-Point, Runx3 transiently formed a complex with pRb and Brd2 and induced Cdkn1a (p21Waf1/Cip1/Sdi1; p21), a key regulator of the R-Point transition. Cyclin D-CDK4/6 promoted dissociation of the pRb-Runx3-Brd2 complex, thus turning off p21 expression. However, cells harboring oncogenic K-Ras maintained the pRb-Runx3-Brd2 complex and p21 expression even after introduction of Cyclin D1. Thus, Runx3 plays a critical role in R-Point regulation and defense against cellular transformation.
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Abstract A43: RUNX3 and pRB form a complex and regulate Restriction-Point commitment
RAS Regulation, 2014Co-Authors: Xin-zi Chi, Jung-won Lee, You-soub Lee, You Mie Lee, Yoshiaki Ito, Suk-chul BaeAbstract:Throughout the cell cycle, a cell monitors cumulative exposure to specific signals over time and makes a critical decision to pass through the Restriction (R) Point. Although the R-Point decision is fundamental to normal differentiation and G1-S transition, the mechanism for the decision has been poorly understood. RUNX3 functions as a tumor suppressor and is frequently inactivated by DNA hyper-methylation in the stomach, bladder, colon, and lung. Recently, we have shown that adenovirus-Cre infected LSL-K-RasG12D mice induced lung adenocarcinoma (ADC) in prolonged latency (median survival, 220 days) and Runx3f/f/LSL-K-RasG12D mice rapidly induced ADC (median survival, 79 days), indicating a strong tumor suppressor activity of Runx3 against oncogenic K-Ras (Lee et., 2013. Cancer Cell. 2013 Vol. 24, pp 603-616). In the present study, we found that targeted disruption of Runx3 results in a delay in pRB phosphorylation and cell cycle progression through the R-Point in mouse embryonic fibroblast cells. During the R-Point interval, RUNX3 transiently formed a complex with pRB and BRD2 and induced Cdkn1a (p21Waf1/Cip1/Sdi1; p21). The complex was dissociated when by Cyclin D1 which was induced 4 hours after serum stimulation and p21 expression was turned off. However, oncogenic K-Ras maintained the complex. These results identify the series of molecular events associated with R-Point commitment and provide an insight into the relationship between R-Point commitment and oncogene surveillance. Note: This abstract was not presented at the conference. Citation Format: Xin-Zi Chi, You-Soub Lee, Jung-Won Lee, You-Mie Lee, Yoshiaki Ito, Suk-Chul Bae. RUNX3 and pRB form a complex and regulate Restriction-Point commitment. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A43. doi: 10.1158/1557-3125.RASONC14-A43
Anders Zetterberg - One of the best experts on this subject based on the ideXlab platform.
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Single cell analysis of G1 check Points-the relationship between the Restriction Point and phosphorylation of pRb.
Experimental cell research, 2005Co-Authors: Hanna-stina Martinsson, Maria Starborg, Fredrik Erlandsson, Anders ZetterbergAbstract:Single cell analysis allows high resolution investigation of temporal relationships between transition events in G1. It has been suggested that phosphorylation of the retinoblastoma tumor suppressor protein (pRb) is the molecular mechanism behind passage through the Restriction Point (R). We performed a detailed single cell study of the temporal relationship between R and pRb phosphorylation in human fibroblasts using time lapse video-microscopy combined with immunocytochemistry. Four principally different criteria for pRb phosphorylation were used, namely (i) phosphorylation of residues Ser795 and Ser780, (ii) degree of pRb-association with the nuclear structure, a property that is closely related with pRb phosphorylation status, (iii) release of the transcription factor E2F-1 from pRb, and (iv) accumulation of cyclin E, which is dependent on phosphorylation of pRb. The analyses of individual cells revealed that passage through R preceded phosphorylation of pRb, which occurs in a gradually increasing proportion of cells in late G1. Our data clearly suggest that pRb phosphorylation is not the molecular mechanism behind the passage through R. The Restriction Point and phosphorylation of pRb thus seem to represent two separate check Point in G1.
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Changes in cell shape and anchorage in relation to the Restriction Point.
Journal of cellular physiology, 2005Co-Authors: Hanna-stina Martinsson, Peter Zickert, Olle Larsson, Maria Starborg, Anders ZetterbergAbstract:The Restriction Point (R) separates the G1 phase of continuously cycling cells into two functionally different parts. The first part, G1-pm, represents the growth factor dependent post-mitotic interval from mitosis to R, which is of constant length (3–4 h). The second part, G1-ps, represents the growth factor independent, pre-S phase interval of G1 that lasts from R to S and that varies in time from 1 to 10 h. G1-pm cells rapidly exit (within 1 h) from the cell cycle and enter G0 as a response to serum withdrawal. The finding that R occurs at a set time after mitosis indicates that R may be related to the metabolic and/or structural changes that the cell underwent during the previous mitosis. We have recently shown that phosphorylation of the retinoblastoma tumor suppressor protein (pRb) is not the molecular mechanism behind R, as has been suggested previously. Here, we present an alternative explanation for R. In the present study, we applied a single cell approach using time-lapse analysis, which revealed that upon serum starvation the G1-pm cells rapidly underwent a transient change in cell shape from flat to spherical before exiting to G0. Platelet derived growth factor (PDGF) counteracted this change in shape and also prevented exit to G0 to the same extent. Furthermore epidermal growth factor (EGF) and insulin like growth factor (IGF-1), which only partially counteracted this change, only partially counteracts exit to G0. These data clearly indicate a direct link between change in cell shape and exit to G0 in G1-cells that have not passed R. © 2004 Wiley-Liss, Inc.
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accumulation of cyclin e is not a prerequisite for passage through the Restriction Point
Molecular and Cellular Biology, 2001Co-Authors: Susanna V Ekholm, Peter Zickert, Steven I Reed, Anders ZetterbergAbstract:In the eukaryotic cell cycle, a reversible growth arrest can be induced in the G1 phase if cells are deprived of growth factors or allowed to grow to confluency (10, 49, 62, 73, 74). Temin (61) showed that chicken cells become independent of external mitogenic growth factors during G1 several hours before entry into S phase. The term Restriction Point (R) was introduced by Pardee (48) to define the Point in G1 after which cells can complete a division cycle independently of mitogenic signals (49). We have previously determined the exact position of R in G1 and its relationship to the previous mitosis and subsequent S phase with time-lapse cinematography (TLC) analysis of mouse and human cells (33, 74, 75, 76, 77). Time-lapse recordings of cells in culture enable analysis of individual cells of an unperturbed, asynchronously growing population. This method is a powerful tool for detailed kinetic analysis of transition events in the cell cycle because, unlike synchronization procedures, it addresses the problem of intercellular variability in cell cycle times, particularly G1 variability. Previous studies using TLC analysis revealed that the G1 phase in cycling cells is separated into two functionally different intervals. During the first part of G1, the G1-pm (postmitosis) period, cell cycle progression is highly dependent on the continuous presence of serum growth factors and on a high rate of protein synthesis. If growth factors are removed from the medium or if protein synthesis is even only moderately inhibited during this period, cells will rapidly (within 30 to 60 min) leave the cell cycle and enter a quiescent state (G0). The G1-pm period has a constant duration of 3 to 4 h in all of the cells studied so far (74, 75, 76). The transition from growth factor-dependent progression to growth factor-independent progression represents passage through R. The part of G1 that follows R, known as the G1-ps (pre-DNA-synthetic) period, is highly variable in duration. Some G1-ps cells initiate DNA replication immediately after passage through R, while others may spend up to 20 h in G1-ps before entering S. This variability in the length of time between R and S implies that even though passage through R is necessary for further progression through the cell cycle, other regulatory events must be completed during G1-ps in order for cells to enter S phase. The cyclins and their catalytic subunits, the cyclin-dependent kinases (Cdks), control cell cycle progression by regulating events that drive the transitions between cell cycle phases. Cyclins were first identified in clam and sea urchin embryos, where they were observed to accumulate during interphase and to be degraded during mitosis (16). Based on homology to invertebrate and frog embryonic cyclins, human A- and B-type cyclins, essential for progression through S, G2, and M phase, were the first human cyclins to be identified (50, 64). Subsequently, the human G1 cyclins, the D-type cyclins and cyclin E, were identified functionally by screening of human cDNA libraries for sequences that could complement G1 cyclin mutations in Saccharomyces cerevisiae (30, 36, 69). The gene for cyclin D1 is induced in response to mitogenic signals as an early-response gene during the transition from G0 to G1 phase and is associated with the catalytic partner Cdk4 or Cdk6. Cyclin E shows a periodic pattern of expression with accumulation in late G1 and downregulation in S (11, 31, 36; reviewed in reference 54). Cyclin E transcription is activated when the retinoblastoma tumor supressor protein (pRb) is hyperphosphorylated and no longer exerts repression of the cyclin E promoter via E2F-DP transcription factor complexes (see below). Consistent with this, a number of putative E2F binding sites have been identified in the cyclin E promoter (19). E2F-mediated repression was first suggested by experiments showing that the combined mutation of two different E2F sites in the human cyclin E promoter leads to partial derepression of the promoter in G1 (45). Recently, a variant E2F-binding site was found to mediate transcriptional repression by binding of a large E2F4-pRb-containing repressor complex (34, 78). Cyclin E associates specifically with Cdk2, and a number of investigations have demonstrated a requirement for cyclin E-cdk2 activity for the initiation of DNA replication (24, 32, 47). Cyclin E is subjected to ubiquitin-dependent degradation during S phase (7, 58, 68). Many of the molecular components that are involved in passage through G1 have been identified, but the molecular mechanism underlying R Point control still remains to be elucidated. Passage through the R Point and phosphorylation-inactivation of pRb have been observed to occur roughly during the same time period in cells entering the cell cycle from G0, but the exact functional relationship between the two events is still unknown. In early G1 phase, pRb is present in an active, hypophosphorylated form, where it is believed to inhibit cell cycle progression by binding to regulatory proteins, including members of the E2F family of transcription factors. Binding of pRb to E2F has been shown to inhibit the transactivation of E2F-dependent genes that are required for cell cycle progression (5, 21, 42). The association between pRb and E2F, as well as other regulatory targets, has been shown to be governed by phosphorylation. pRb is phosphorylated at multiple sites as Cdk activity increases during G1 phase. Hyperphosphorylated pRb first appears during late G1 phase. Although several Cdks have been implicated in pRb phosphorylation in vitro (1, 17, 40, 41), the precise mechanism by which pRb is phosphorylated in vivo is still unclear. By ectopically expressing cyclin D1 or E during early G1, it was demonstrated that expression of either cyclin shortens the G1 phase in rat embryonic fibroblasts but only cyclin D1 expression leads to premature pRb phosphorylation (55). Other evidence suggests that pRb is phosphorylated by both cyclin D- and E-dependent kinases in a sequential manner to achieve hyperphosphorylation (8, 20, 39, 70). Therefore, it has been proposed that the accumulation of either D-type cyclins or cyclin E and the concomitant hyperphosphorylation/inactivation of pRb constitute progression through the R Point (9, 53, 77). Consistent with this idea, it has been demonstrated that passage through the R Point is dependent on the accumulation of a labile protein (48), a characteristic of both D-type cyclins and cyclin E (11, 36, 37, 47). The aim of the present study was to perform a detailed analysis of cyclin E expression in relation to passage through R and entry into S phase, in order to determine whether cyclin E could be the labile R-associated protein. In order to determine the exact timing of cyclin E accumulation and downregulation, we carried out an analysis of individual cells which allowed us to consider the variability of cell behavior within the population. We found that cells younger than 3.5 h after mitosis, i.e., cells that had not yet passed R, were negative for cyclin E accumulation. After passage through R, cyclin E begins to accumulate as a cell approaches S, however, with a high degree of temporal variability. These data indicate that passage through R cannot be dependent on the accumulation of cyclin E, as has been proposed, and suggest that since R occurs prior to the accumulation of cyclin E, passage through R may be a prerequisite for cyclin E accumulation. Furthermore, the temporal variability of cyclin E accumulation after passage through R suggests that another late-G1 event(s) controls the precise timing of cyclin E accumulation.
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WHAT IS THE Restriction Point
Current opinion in cell biology, 1995Co-Authors: Anders Zetterberg, Olle Larsson, Klas G. WimanAbstract:Abstract The Restriction Point (R) separates two functionally different parts of G1 in continuously cycling cells. G1-pm represents the postmitotic interval of G1 that lasts from mitosis to R. G1-ps represents the pre S phase interval of G1 that lasts from R to S. G1-pm is remarkably constant in length (its duration is about three hours) in the different cell types studied so far. G1-ps, however, varies considerably, indicating that entry into S is not directly followed after passage through R. Progression through G1-pm requires continuous stimulation by mitogenic signals (e.g. growth factors) and a high rate of protein synthesis. Interruption of the mitogenic signals or moderate inhibition of protein synthesis leads to a rapid exit from the cell cycle to G0 in normal (untransformed) cells. Upon restimulation with mitogenic signals, the cell returns to the same Point in G1-pm from which it left the cell cycle. Thus the cell seems to have a memory for how far it has advanced through G1-pm, suggesting that a continuous structural alteration, for example chromatin decondensation, takes place in G1. The molecular background to transition from growth factor dependence in G1-pm to growth factor independence in G1-ps (a switch which represents commitment to a new cell cycle and passage through R) is still not fully understood. Cyclin-dependent kinase (cdk)-mediated hyperphosphorylation of the retinoblastoma protein (Rb), and concomitant liberation (and activation) of members of the E2F family of transcription factors, are probably important aspects of R control in normal cells. A key component here could be cdk2 activity which is controlled by cyclin E. When cdk2 activity starts to increase rapidly in G1, due to activation of a positive feedback loop, it reaches a critical level above which cdk inhibitors (CKIs) such as p21 and p27 are outweighed; the cell has then become independent of mitogenic and inhibitory signals and is committed to a new cell cycle. However, other components are probably also involved in R control. For instance, a ‘cryptic’ R (a G1-pm-like state) can be induced even in tumour cells that do not respond to growth factor starvation or protein synthesis inhibitors, and are therefore probably defective in the cdk-Rb-E2F pathway. Possibly, a certain degree of chromatin decondensation has to take place after mitosis in order to allow transcription of, for example, the cyclin E gene or other critical E2F targets. Although the molecular basis for Restriction Point control still remains unclear, we can expect rapid progress in this important field over the next few years.
Yuval Yung - One of the best experts on this subject based on the ideXlab platform.
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a reciprocal relationship between rb and skp2 implications for Restriction Point control signal transduction to the cell cycle and cancer
Cell Cycle, 2008Co-Authors: Richard K Assoian, Yuval YungAbstract:The identification of Skp2 as a direct E2F target has allowed us to document the existence of a positive feedback loop comprised of Rb-E2F, Skp2, p27 and cyclin E-cdk2. We have termed this set of regulatory molecules the Skp2 autoinduction loop. Interference with this loop selectively regulates cell cycle progression through the Restriction Point. We describe here how the Skp2 autoinduction loop may interact with other regulatory controls on the Restriction Point, and how the reciprocal relationship between Rb and Skp2 may affect signal transduction to the cell cycle and thinking about the role of Skp2 overexpression in cancers.
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A Skp2 autoinduction loop and Restriction Point control.
The Journal of cell biology, 2007Co-Authors: Yuval Yung, James M Roberts, Janice L. Walker, Richard K AssoianAbstract:We describe a self-amplifying feedback loop that autoinduces Skp2 during G1 phase progression. This loop, which contains Skp2 itself, p27kip1 (p27), cyclin E–cyclin dependent kinase 2, and the retinoblastoma protein, is closed through a newly identified, conserved E2F site in the Skp2 promoter. Interference with the loop, by knockin of a Skp2-resistant p27 mutant (p27T187A), delays passage through the Restriction Point but does not interfere with S phase entry under continuous serum stimulation. Skp2 knock down inhibits S phase entry in nontransformed mouse embryonic fibroblasts but not in human papilloma virus–E7 expressing fibroblasts. We propose that the essential role for Skp2-dependent degradation of p27 is in the formation of an autoinduction loop that selectively controls the transition to mitogen-independence, and that Skp2-dependent proteolysis may be dispensable when pocket proteins are constitutively inactivated.
Richard K Assoian - One of the best experts on this subject based on the ideXlab platform.
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a reciprocal relationship between rb and skp2 implications for Restriction Point control signal transduction to the cell cycle and cancer
Cell Cycle, 2008Co-Authors: Richard K Assoian, Yuval YungAbstract:The identification of Skp2 as a direct E2F target has allowed us to document the existence of a positive feedback loop comprised of Rb-E2F, Skp2, p27 and cyclin E-cdk2. We have termed this set of regulatory molecules the Skp2 autoinduction loop. Interference with this loop selectively regulates cell cycle progression through the Restriction Point. We describe here how the Skp2 autoinduction loop may interact with other regulatory controls on the Restriction Point, and how the reciprocal relationship between Rb and Skp2 may affect signal transduction to the cell cycle and thinking about the role of Skp2 overexpression in cancers.
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A Skp2 autoinduction loop and Restriction Point control.
The Journal of cell biology, 2007Co-Authors: Yuval Yung, James M Roberts, Janice L. Walker, Richard K AssoianAbstract:We describe a self-amplifying feedback loop that autoinduces Skp2 during G1 phase progression. This loop, which contains Skp2 itself, p27kip1 (p27), cyclin E–cyclin dependent kinase 2, and the retinoblastoma protein, is closed through a newly identified, conserved E2F site in the Skp2 promoter. Interference with the loop, by knockin of a Skp2-resistant p27 mutant (p27T187A), delays passage through the Restriction Point but does not interfere with S phase entry under continuous serum stimulation. Skp2 knock down inhibits S phase entry in nontransformed mouse embryonic fibroblasts but not in human papilloma virus–E7 expressing fibroblasts. We propose that the essential role for Skp2-dependent degradation of p27 is in the formation of an autoinduction loop that selectively controls the transition to mitogen-independence, and that Skp2-dependent proteolysis may be dispensable when pocket proteins are constitutively inactivated.
Hein Te Riele - One of the best experts on this subject based on the ideXlab platform.
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In vivo significance of the G2 Restriction Point.
Cancer research, 2007Co-Authors: Floris Foijer, Elly Delzenne-goette, Marleen Dekker, Hein Te RieleAbstract:Loss of activity of the retinoblastoma pathway is a common event in human cancer. Mouse models have revealed that tumorigenesis by loss of Rb was accelerated by concomitant loss of the cell cycle inhibitor p27KIP1. This has been attributed to reduced apoptosis and weakening of the G1 checkPoint. However, the role of p27KIP1 in a recently identified G2 Restriction Point may offer an alternative explanation for this synergy. Here, we have investigated the significance of the G2 Restriction Point in Rb-deficient pituitaries. We show that Rb loss in the pituitary gland activated the G2 Restriction Point, as evidenced by the appearance of cyclin B1-p27KIP1 complexes. Somewhat unexpectedly, these complexes remained present in Rb-deficient tumors. These results indicate that the G2 Restriction Point does operate in vivo. However, in the pituitary gland, this mechanism seems to retard rather than to prevent tumor growth.
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Restriction beyond the Restriction Point: mitogen requirement for G2 passage.
Cell division, 2006Co-Authors: Floris Foijer, Hein Te RieleAbstract:Cell proliferation is dependent on mitogenic signalling. When absent, normal cells cannot pass the G1 Restriction Point, resulting in cell cycle arrest. Passage through the G1 Restriction Point involves inactivation of the retinoblastoma protein family. Consequently, loss of the retinoblastoma protein family leads to loss of the G1 Restriction Point. Recent work in our lab has revealed that cells possess yet another mechanism that restricts proliferation in the absence of mitogens: arrest in the G2 phase of the cell cycle. Here, we discuss the similarities and differences between these Restriction Points and the roles of cyclin-dependent kinase inhibitors (CKIs) herein.
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Mitogen requirement for cell cycle progression in the absence of pocket protein activity
Cancer cell, 2005Co-Authors: Floris Foijer, Rob M. F. Wolthuis, Valerie D. Doodeman, René H. Medema, Hein Te RieleAbstract:Primary mouse embryonic fibroblasts lacking expression of all three retinoblastoma protein family members (TKO MEFs) have lost the G1 Restriction Point. However, in the absence of mitogens these cells become highly sensitive to apoptosis. Here, we show that TKO MEFs that survive serum depletion pass G1 but completely arrest in G2. p21CIP1 and p27KIP1 inhibit Cyclin A-Cdk2 activity and sequester Cyclin B1-Cdk1 in inactive complexes in the nucleus. This response is alleviated by mitogen restimulation or inactivation of p53. Thus, our results disclose a cell cycle arrest mechanism in G2 that restricts the proliferative capacity of mitogen-deprived cells that have lost the G1 Restriction Point. The involvement of p53 provides a rationale for the synergism between loss of Rb and p53 in tumorigenesis.